PROJECT SUMMARY/ABSTRACT Renal cell carcinoma (RCC) is a lethal disease whose incidence is on the rise. It is categorized into various subtypes, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. Current targeted molecular strategies, including tyrosine kinase inhibitors (TKIs), have resulted in a doubling of progression-free survival and significant gains in overall survival in ccRCC patients. Despite the therapeutic progress, complete and durable responses have been noted in only a few cases. Moreover, about one quarter of the ccRCC patients are primarily refractory to treatment with TKIs. It is well established that TKIs have a direct cytotoxic effect on tumor cells in addition to the inhibition of tumor angiogenesis. We have applied CRISPR/Cas9 based high- throughput loss-of-function screening to identify new genes involved in the resistance to sunitinib, one of the most commonly used TKIs given to RCC patients. This screen identified farnesyltransferase (FTase)- dependent proteins regulating endosomal/lysosomal formation as important factors contributing to sunitinib resistance. Several studies indicate lysosomal sequestration as a novel mechanism of drug resistance. In addition, the farnesylation of Rheb is required for activation of mTORC1 signaling. Importantly, inhibition of mTORC1 reinstates sensitivity to TKIs in tumor cells of various origins. Excitingly, treatment with a clinically relevant FTase inhibitor (FTIs), lonafarnib, completely reinstates the sensitivity of RCC cells to sunitinib both in vitro and in vivo. In addition, combinatorial treatment with sunitnib + lonafarnib was well tolerated in vivo, highlighting the potential promise in the clinic. The proposed studies will test a novel hypothesis that lonafarnib enhances the antitumor activity of sunitinib, at least in part, through two potential mechanisms: (1) dysregulation of lysosomal sequestration of sunitinib, and (2) suppression of Rheb-mediated mTORC1 activation. To test our hypothesis and to validate the therapeutic value of combined treatment with lonafarnib and sunitinib, we propose the following Specific Aims: (1) To identify mechanisms underlying the synergistic antitumor activity of TKIs and FTIs; (2) To evaluate the antitumor efficacy of combined sunitinib and lonafarnib treatment using direct human-to-mouse xenograft model of ccRCC.